Many intracellular pathogens survive in host organisms by coordinately regulating the expression of a wide spectrum of genes in response to their surroundings. This adaptation includes not only metabolic and physiological adjustments to new nutritional requirements, but also the synthesis of proteins necessary to circumvent the host organism's anti-microbial arsenal. For example, bacteria survive in phagocytes by expressing certain genes in response to the phagocytic environment. Since a microbe's ability to survive in the host correlates with its ability to cause disease, the identification of genes that are preferentially transcribed in the in vivo environment of the host is central to our understanding of how pathogenic organisms mount a successful infection. For further information on the relationship between gene expression and pathogen survival in hosts, see the articles by Mekalanos in J. Bacteriol. 174:1 (1992), Mahan et al. in Escherichia coli and Salmonella typhimurium F. C. Neidhart, Ed. (ASM Press, Washington D.C., 1996), vol. II, pp. 2803, Fields et al. in PNAS USA 83:5189 (1986), and Horwitz in J. Exp. Med. 166:1310 (1987).
Recently, the isolation of preferentially-induced genes has been made possible with the use of sophisticated promoter traps (e.g. IVET) that are based on conditional auxotrophy complementation or drug resistance. In one IVET approach, various bacterial genome fragments are placed in front of a necessary metabolic gene coupled to a reporter gene. The DNA constructs are inserted into a bacterial strain otherwise lacking the metabolic gene, and the resulting bacteria are used to infect the host organism. Only bacteria expressing the metabolic gene survive in the host organism; consequently, inactive constructs can be eliminated by harvesting only bacteria that survive for some minimum period in the host. At the same time, constitutively active constructs can be eliminated by screening only bacteria which do not express the reporter gene under laboratory conditions. The bacteria selected by such a method contain constructs that are selectively induced only during infection of the host. The genome fragments in such constructs make promising therapeutic targets. For information on IVET see the articles by Mahan et al. in Science 259:686-688 (1993), Mahan et al. in PNAS USA 92:669-673 (1995), Heithoff et al. in PNAS USA 94:934-939 (1997), and Wanget al. in PNAS USA. 93:10434 (1996).
IVET has been limited to bacterial pathogens with tractable genetic systems because of its requirement for high frequencies of homologous recombination and extensive strain manipulation prior to gene selection. In IVET, the library of constructs is made by recombination into the chromosome; consequently, building a representative library can be difficult and labor-intensive. The technique's reliance on conditional auxotrophic complementation or drug resistance limits its use to organisms with particular nutritional requirements or antibiotic sensitivity. Gene fusions that are transcriptionally silent under laboratory conditions must be manually screened, a step which is not only biased but also time-consuming. The use of lacZ or other conventional reporter genes requires the addition of substrates to the bacteria. In addition, the method is limited to the measurement of gene activity on a populational basis, and is sensitive to the bacterial load present within cells and to the effect of microenvironments on enzymatic activity (e.g., lacZ is irreversibly denatured below pH 5.5).